Project Summary/Abstract To date, pancreatic cancer, beyond most other cancers has been categorically associated with the term ?lethal?. This association stems from the fact that pancreatic cancer has a median survival rate of less than 6 months after diagnosis, and a bleak 5-year survival rate of 3-5 percent. The most prevalent form of pancreatic cancer, pancreatic ductal adenocarcinoma (PDAC), demonstrates a particularly aggressive biology with resistance to both conventional and targeted therapeutics, so that by the time a patient receives the diagnosis, the disease has already advanced to an incurable state. Furthermore, significant challenges exist in obtaining tissue from pancreatic cancer patients, making it difficult to study tumors and their pharmacodynamic responses during treatment. These facts highlight the unmet challenge of identifying the lethal cells that survive and thrive even after treatment and are predisposed to recur. Ascertaining the mechanisms that drive the disease and its recurrence can spur the development of new treatment strategies to improve outcomes for these patients. One avenue that could lead to accurate predictive tools, therapeutic targets, and pharmacodynamic biomarker information comes from the analysis of circulating tumor cells (CTCs). For over two decades, studies have shown that tumor cells from primary solid tumors can be detected in the circulation. These CTCs may be precursors to systemic metastases. The detection of CTCs in peripheral blood has been recognized as a potential tool in the diagnosis of cancer and cell metastasis. Furthermore, the relative number of CTCs in the blood appears to be an independent predictor of progression in several types of cancer. However, before instituting CTCs as a reliable biomarker, one must answer fundamental questions regarding their biological and clinical significance. Are all CTCs capable of proliferation, invasion, and metastasis? Are there subpopulations of CTCs that are more aggressive than the rest? If so, do these aggressive CTCs carry specific driver signature and how it is different or similar to primary tumor? Are these cells persistent through therapy and capable of proliferation? Answers to these questions can reveal the earliest cells with metastasis- initiating capability, providing a therapeutic target. Hence, to establish clinical applications of CTCs for personalized therapy in pancreatic and other cancers, there is a compelling need for sensitive, accurate approaches to distinguish metastasis-initiating driver CTCs from essentially ?passenger? CTCs. We will accomplish this goal through a sophisticated biomarker independent microfluidic platform ?labyrinth? that not only enables highly sensitive isolation of CTCs but also provides the novel capability to culture and expand the low numbers of isolated CTCs. Most of the CTC isolation technologies depend on the known surface markers and suffer from low throughput. Moreover, the presence of multiple sub-populations of CTCs (including those of an EMT phenotype) with different metastatic potential requires a method for separating these cells from blood that does not depend on specific antibodies such as EpCAM. In this proposal, we will develop, test and optimize the efficacy of a novel antigen-independent microfluidic platform (the labyrinth) in the isolation, characterization, ex vivo expansion and therapeutic targets testing of pancreatic cancer CTCs. Labyrinth takes advantage of inertial forces in curved geometries to differentially focus cells based on their size to isolate CTCs from whole blood at a high throughput of 2.5mL/min (150mL/hr) in a single step without any aid of a biomarker. The multiple turns (more than 50) ensure focusing of even the smaller cells while traversing through the device, and hence achieve a remarkable separation between WBCs and CTCs resulting in high purity. The unique ability to isolate CTCs in a label free manner at a high throughput, with greater sensitivity and reliably expand few numbers of CTCs opens exciting, new opportunities for biologic, genomic, and functional analyses of CTCs, which we expect will change the paradigm for use of CTCs in clinical oncology.